Rationally designed, synthetic antibody libraries and uses therefor

Abstract

The present invention overcomes the inadequacies inherent in the known methods for generating libraries of antibody-encoding polynucleotides by specifically designing the libraries with directed sequence and length diversity. The libraries are designed to reflect the preimmune repertoire naturally created by the human immune system, with or without DH segments derived from other species, and are based on rational design informed by examination of publicly available databases of antibody sequences.

What is claimed is: 
     
       1. A library comprising synthetic polynucleotides that encode an antibody heavy chain or alternative scaffold containing at least about 10 6  unique antibody CDRH3 amino acid sequences, wherein each of the polynucleotides encoding the at least about 10 6  unique antibody CDRH3 amino acid sequences has an antibody CDRH3 sequence represented by the following formula:
   [X]−[N1]−[DH]−[N2]−[H3−JH], wherein
 
 (i) X is selected from the group consisting of G, D, E, and nothing; 
 (ii) N1 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, and WGT; 
 (iii) DH is all of a non-human germline CDRH3 DH amino acid sequence from a vertebrate species; 
 (iv) N2 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, and WGT; and 
 (v) H3−JH is all of a human CDRH3 H3−JH amino acid sequence, wherein the H3−JH amino acid sequence is selected from the group consisting of AEYFQH (SEQ ID NO: 17), EYFQH (SEQ ID NO: 583), YFQH (SEQ ID NO: 584), FQH, QH, H, YWYFDL (SEQ ID NO: 18), WYFDL (SEQ ID NO: 585), YFDL (SEQ ID NO: 586), FDL, DL, L, AFDV (SEQ ID NO: 19), FDV, DV, V, YFDY (SEQ ID NO: 20), FDY, DY, Y, NWFDS (SEQ ID NO: 21), WFDS (SEQ ID NO: 587), FDS, DS, S, YYYYYGMDV (SEQ ID NO: 22), YYYYGMDV (SEQ ID NO: 588), YYYGMDV (SEQ ID NO: 589), YYGMDV (SEQ ID NO: 590), YGMDV (SEQ ID NO: 591), GMDV (SEQ ID NO: 592), and MDV; 
 wherein the diversity of the polynucleotides encoding the at least about 10 6  unique antibody CDRH3 sequences is created by each of the polynucleotides having a CDRH3 sequence that is different from the CDRH3 sequence of every other polynucleotide; 
 wherein the antibody heavy chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMH1-CDRH1-FRMH2-CDRH2-FRMH3-CDRH3-FRMH4; and 
 wherein the alternative scaffold is selected from the group consisting of fibronectin, the β-sandwich, lipocalin, EETI-IFAGRP, BPTIFLAC1-D1/IT1-D2, thioredoxin, protein A, ankyrin repeats, γB-crystallin/ubiquitin, CTLD3 and (LDLR-A module) 3 . 
 
     
     
       2. The library of  claim 1 , wherein the vertebrate species is selected from the group consisting of  Mus musculus, Camelus  sp.,  Llama  sp.,  Camelidae  sp.,  Raja  sp.,  Ginglymostoma  sp.,  Carcharhinus  sp.,  Heterodontus  sp.,  Hydrolagus  sp.,  Ictalurus  sp.,  Gallus  sp.,  Bos  sp.,  Marmaronetta  sp.,  Aythya  sp.,  Netta  sp.,  Equus  sp.,  Pentalagus  sp.,  Bunolagus  sp.,  Nesolagus  sp.,  Romerolagus  sp.,  Brachylagus  sp.,  Sylvilagus  sp.,  Oryctolagus , sp.,  Poelagus  sp.,  Ovis  sp.,  Sus  sp.,  Gadus  sp.,  Salmo  sp.,  Oncorhynchus  sp,  Macaca  sp.,  Rattus  sp.,  Pan  sp.,  Hexanchus  sp.,  Heptranchias  sp.,  Notorynchus  sp.,  Chlamydoselachus  sp.,  Heterodontus  sp.  Pristiophorus  sp.,  Pliotrema  sp.,  Squatina  sp.,  Carcharia  sp.,  Mitsukurina  sp.,  Lamma  sp.,  Isurus  sp.,  Carcharodon  sp.,  Cetorhinus  sp.,  Alopias  sp.,  Nebrius  sp.,  Stegostoma  sp.,  Orectolobus  sp.,  Eucrossorhinus  sp.,  Sutorectus  sp.,  Chiloscyllium  sp.,  Hemiscyllium  sp.,  Brachaelurus  sp.,  Heteroscyllium  sp.,  Cirrhoscyllium  sp.,  Parascyllium  sp.,  Rhincodon  sp.,  Apristurus  sp.,  Atelomycterus  sp.,  Cephaloscyllium  sp.,  Cephalurus  sp.,  Dichichthys  sp.,  Gallus  sp.,  Halaelurus  sp.,  Haploblepharus  sp.,  Parmaturus  sp.,  Pentanchus  sp.,  Poroderna  sp.,  Schroederichthys  sp.,  Scyliorhinus  sp.,  Pseudotriakis  sp.,  Scylliogaleus  sp.,  Furgaleus  sp.,  Hemitriakis  sp.,  Mustelus  sp.,  Triakis  sp.,  Iago  sp.,  Galeorhinus  sp.,  Hypogaleus  sp.,  Chaenogaleus  sp.,  Hemigaleus  sp.,  Paragaleus  sp.,  Galeocerdo  sp.,  Prionace  sp.,  Sciolodon  sp.,  Loxodon  sp.,  Rhizoprionodon  sp.,  Aprionodon  sp.,  Negaprion  sp.,  Hypoprion  sp.,  Carcharhinus  sp.,  Isogomphodon  sp.,  Triaenodon  sp.,  Sphyrna  sp.,  Echinorhinus  sp.,  Oxynotus  sp.,  Squalus  sp.,  Centroscyllium  sp.,  Etmopterus  sp.,  Centrophorus  sp.,  Cirrhigaleus  sp.,  Deania  sp.,  Centroscymnus  sp.,  Scymnodon  sp.,  Dalatias  sp.,  Euprotomicrus  sp.,  Isistius  sp.,  Squaliolus  sp.,  Heteroscymnoides  sp.,  Somniosus  sp. and  Megachasma  sp. 
     
     
       3. The library of  claim 1 , wherein the CDRH3 amino acid sequences are expressed as part of full-length heavy chains. 
     
     
       4. A library of vectors comprising the polynucleotide library of  claim 1 . 
     
     
       5. A population of cells comprising the vectors of  claim 4 . 
     
     
       6. The cells of  claim 5 , wherein the cells are yeast cells. 
     
     
       7. The cells of  claim 5 , wherein the cells are  S. cerevisiae.    
     
     
       8. A kit comprising a library comprising synthetic polynucleotides that encode an antibody heavy chain or alternative scaffold containing at least about 10 6  unique antibody CDRH3 amino acid sequences, wherein each of the polynucleotides encoding the at least about 10 6  unique-antibody CDRH3 amino acid sequences has an antibody CDRH3 sequence represented by the following formula:
   [X]−[N1]−[DH]−[N2]−[H3−JH], wherein
 
 (i) X is selected from the group consisting of G, D, E, and nothing; 
 (ii) N1 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, and WGT; 
 (iii) DH is all of a non-human germline CDRH3 DH amino acid sequence from a vertebrate species; 
 (iv) N2 is an amino acid sequence selected from the group consisting of G, P, R, A, S, L, T, V, GG, GP, GR, GA, GS, GL, GT, GV, PG, RG, AG, SG, LG, TG, VG, PP, PR, PA, PS, PL, PT, PV, RP, AP, SP, LP, TP, VP, GGG, GPG, GRG, GAG, GSG, GLG, GTG, GVG, PGG, RGG, AGG, SGG, LGG, TGG, VGG, GGP, GGR, GGA, GGS, GGL, GGT, GGV, D, E, F, H, I, K, M, Q, W, Y, AR, AS, AT, AY, DL, DT, EA, EK, FH, FS, HL, HW, IS, KV, LD, LE, LR, LS, LT, NR, NT, QE, QL, QT, RA, RD, RE, RF, RH, RL, RR, RS, RV, SA, SD, SE, SF, SI, SK, SL, SQ, SR, SS, ST, SV, TA, TR, TS, TT, TW, VD, VS, WS, YS, AAE, AYH, DTL, EKR, ISR, NTP, PKS, PRP, PTA, PTQ, REL, RPL, SAA, SAL, SGL, SSE, TGL, and WGT; 
 (v) H3−JH is all of a human CDRH3 H3−JH amino acid sequence, wherein the H3−JH amino acid sequence is selected from the group consisting of AEYFQH (SEQ ID NO: 17), EYFQH (SEQ ID NO: 583), YFQH (SEQ ID NO: 584), FQH, QH, H, YWYFDL (SEQ ID NO: 18), WYFDL (SEQ ID NO: 585), YFDL (SEQ ID NO: 586), FDL, DL, L, AFDV (SEQ ID NO: 19), FDV, DV, V, YFDY (SEQ ID NO: 20), FDY, DY, Y, NWFDS (SEQ ID NO: 21), WFDS (SEQ ID NO: 587), FDS, DS, S, YYYYYGMDV (SEQ ID NO: 22), YYYYGMDV (SEQ ID NO: 588), YYYGMDV (SEQ ID NO: 589), YYGMDV (SEQ ID NO: 590), YGMDV (SEQ ID NO: 591), GMDV (SEQ ID NO: 592), and MDV; 
 wherein the diversity of the polynucleotides encoding the at least about 10 6  unique antibody CDRH3 sequences is created by each of the polynucleotides having a CDRH3 sequence that is different from the CDRH3 sequence of every other polynucleotide; 
 wherein the antibody heavy chain is a variable domain with framework (FRM) and complementary determining regions (CDR) comprising FRMH1-CDRH1-FRMH2-CDRH2-FRMH3-CDRH3-FRMH4; and 
 wherein the alternative scaffold is selected from the group consisting of fibronectin, the β-sandwich, lipocalin, EETI-IFAGRP, BPTIFLAC1-D1/IT1-D2, thioredoxin, protein A, ankyrin repeats, γB-crystallin/ubiquitin, CTLD3 and (LDLR-A module) 3 .